1. Field of the Invention
The invention relates generally to biosensors and methods of detecting and quantifying specific proteins, factors and chemical ligands, in particular sequence-specific nucleic acid binding factors and their coregulators, by changes in luminescence signal intensity or by detection of a detectable label. The invention is used in any application where the determination of the activity of a nucleic acid binding factor or of a coregulator thereof is desired.
2. Description of the Related Art
The ability to detect and quantify specific chemicals and chemical moieties, especially nucleic acid binding factors and their coregulators is of great importance in basic research and in clinical applications. Determination of the level of a specific protein or other biomolecule is one of the most useful and important experimental procedures in biomedical research and molecular diagnostics. Cellular levels of specific proteins or cofactors are commonly used as diagnostic markers for many diseases.
Protein-nucleic acid interactions are an extremely important and physiologically relevant type of macromolecular contact found in the cell. Many proteins that play an important role in regulating many processes in prokaryotic cells, eukaryotic cells, and viruses possess natural sequence-specific nucleic acid binding activity. These proteins include transcription factors, chromatin remodeling factors and DNA maintenance enzymes. For a review of nucleic acid binding factors, see Benjamin Lewin, Genes VII, Oxford University Press, New York, 2000, which is herein incorporated by reference.
Transcription factors bind to specific cognate nucleic acid elements, which include promoters, enhancers and silencer elements. They may be activators, repressors or both, depending on the cellular context, whose levels are important for regulation of gene expression. Thus, many of these proteins are important in disease development and disease diagnosis. For example, several transcription factors, which when overexpressed or inappropriately expressed, are oncogenes. These oncogenic transcription factors include myc, myb, fos, jun, rel and erb. Another cancer related transcription factor, p53, is involved in development of many cancers (Ko, L. L., and Prives, C. Genes Dev. 10, 1054-1072, 1996).
Chromatin remodeling factors are also important for the regulation of gene expression. Generally, regions of highly condensed chromatin, called heterochromatin, contain genes that are not actively transcribed, whereas regions of loose or non-condensed chromatin, called euchromatin, contain genes that are actively transcribed. During cellular differentiation, cancerous transformation and normal physiological homeostasis, chromatin may be remodeled. That is, some chromosomal regions become inaccessible to transcription factors and RNA polymerase, whereas other regions become accessible. Several nucleic acid binding factors are involved in this dynamic process including nucleosome proteins (e.g., histones), histone acetyltransferases, histone deacetylases, amino acid methyltransferases, DNA methyltransferases, nucleoplasmins, HMG proteins, repressor complex proteins, polycomb-related factors and trithorax-related factors.
DNA maintenance enzymes are nucleic acid binding factors necessary for the repair of damaged DNA, faithful replication of DNA and exchange of genetic information during recombination. Several types of cancer and other disease syndromes are the result of defective DNA maintenance enzymes. For example, Xeroderma pigmentosum, a horrific genetic disease whereby the sufferer is predisposed to skin cancer, is due to defective nucleotide-excision repair enzymes. Hereditary non-polyposis colorectal cancer is caused in large part by defective mismatch repair enzymes. Some forms of hereditary breast cancers are due to defective homologous recombination enzymes. For a review of genome maintenance systems and their role in cancer, see Hoeijmakers, J. H. J., Nature 411, 366374, 2001, which is herein incorporated by reference. Thus, there is a significant interest in convenient and accurate methods for detecting, monitoring and/or quantifying nucleic acid binding activity of nucleic acid binding factors and their coregulators.
The most common approaches taken to detect proteins exhibiting sequence-specific nucleic acid binding activity are gel shift assays and various nucleic acid footprinting assays (Fried, M. G., and Crothers, D. M. Nucleic Acids Res. 9, 6505-6525, 1981; Galas, D. J., and Schmitz, A. Nucleic Acid Res. 5, 3157-3170, 1978). These methods are laborious and time-consuming procedures, which typically involve the use of dangerous and expensive radioisotopes. Furthermore, these methods are not generally adaptable to high-throughput assay formats. Different fluorescence based methodologies for detecting and studying nucleic acid binding factors have been developed to overcome the deficiencies of gel shift and nucleic acid footprinting assays.
Detection of molecules by fluorescence has several advantages compared to alternative detection methods. Fluorescence provides an unmatched sensitivity of detection, as demonstrated by the detection of single molecules using fluorescence (Weiss, S. Science 283, 1676-1683, 1999). Detection of fluorescence, changes in fluorescence intensity or changes in emission spectra can be easily achieved by the selection of specific wavelengths of excitation and emission. Fluorescence provides a real-time signal allowing real-time monitoring of processes and real-time cellular imaging by microscopy (see Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Kluwer AcademiclPlenum Press, New York, 1999, which is herein incorporated by reference). Additionally, well-established methods and instrumentation for high-throughput detection of fluorescence signals exist in the art.
Current methods for detecting nucleic acid binding factors in solution using fluorescence rely on one of the following phenomena: (i) a change in the fluorescence intensity of a fluorochrome (also called a fluorophore or a fluorescent probe or label), which is present either on the protein or on the nucleic acid, as a result of the perturbation of the microenvironment of the probe upon protein-nucleic acid complex formation; (ii) a change of fluorescence polarization of the fluorochrome, which is present either on the protein or on the nucleic acid, as a result of an increase in the molecular size of the protein-nucleic acid complex relative to the unbound nucleic acid or protein molecules; and (iii) resonance energy transfer between one fluorochrome present in nucleic acid and another fluorochrome or fluorescence quencher present in a protein as a result the proximity between nucleic acid and the protein in protein-nucleic acid complex. For a review on methods of detecting fluorescence signal detection, see Hill, J. J., and Royer, C. A. Methods in Enzymol. 278, 390-416 (1997), which is herein incorporated by reference. Examples of the application of a change in fluorescence intensity of a fluorchrome to the detection of protein-nucleic acid complexes using fluorochromes attached to the protein or the nucleic acid can be found in the following technical literature, which are herein incorporated by reference (Sha, M., FerreD'Amare, Burley, S. K., and Goss, D. J. J. Biol. Chem. 270, 19325-19329, 1995; Reedstrom, R. J., Brown, M. P., Grillo, A., Roen, D, and Royer, C. A. J. Mol. Biol. 273, 572-585, 1997; Erickson, G. H, and Daksis, J. WO 00140753).
Another type of fluorescence-based detection assay, called fluorescence polarization, has also been used for the detection of protein-nucleic acid complex formation (see Heyduk, T., and Lee, J. C. Proc. Natl. Acad. Sci USA 87, 1744-1748, 1990, which is herein incorporated by reference). The physical basis of this approach is that the fluorescence polarization signal of a macromolecule labeled with a fluorochrome depends on the size of the macromolecule (Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Press, New York, 1999, herein incorporated by reference). Hence, upon the formation of a protein-nucleic acid complex from the protein and nucleic acid components, a larger molecular entity is created, which has an altered fluorescence signature. The use of fluorescence polarization to detect protein-nucleic acid complexes is described in Royer (1998, U.S. Pat. No. 5,756,292), which is herein incorporated by reference.
A third fluorescence-based assay for the detection of the protein-nucleic acid complex formation is fluorescence resonance energy transfer (FRET) (Stryer, L. Ann. Rev. Biochem. 47, 819-846, 1978, which is herein incorporated by reference). FRET is based upon the transfer of emitted light energy from a fluorochrome (fluorescent donor) to an acceptor molecule (fluorescent acceptor), which may also be a fluorochrome. As used in the detection of nucleic acid-protein binding events, the FRET assay is based on the increased proximity between a fluorochrome attached to the DNA binding protein and the fluorochrome attached to the cognate DNA binding element when a binding event has occurred. Several published reports illustrate the use of this approach to detect and study protein-nucleic acid interactions (see Kane, S. A., Fleener, C. A., Zhang, Y. S., Davis, L. J., Musselman, A. L., and Huang, P. S. Anal. Biochem. 278, 29-39, 2000, which is herein incorporated by reference).
In summary, luminescence or fluorescence-based assay systems are attractive tools for detecting nucleic acid binding proteins. The inventor describes herein a general, inexpensive, simple, multicolor or single color fluorescence, luminescence, radiographic, gravimetric or calorimetric method for detecting sequence specific nucleic acid binding factors or coregulators thereof, which are compatible with high-throughput detection formats.